This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. ABSTRACT: We are extending our work on the ryanodine receptor in skeletal muscle to in-situ observation, with the hope of visualizing dihydropyridine receptors (the voltage sensors that mediate the electrical impulse and the release of calcium that causes contraction). In the single-particle ryanodine receptor work, and the work on isolated triad junctions, done so far in Dr. Wagenknecht's lab, the dihydropyridine receptors were either removed or disrupted during isolation. We are collaborating with Dr. Clara Franzini-Armstrong of the University of Pennsylvania, who is a leader in the study of the excitation-contraction apparatus. With the benefit of her long experience in preparing muscle tissue for optimal visualization of the triad junction in situ, we are developing methods for high-pressure freezing muscle tissue in the proper orientation for observation of the dihydropyridine receptors. In the previous reporting period, Dr. Franzini-Armstrong visited the Resource and presented a seminar to describe the project. She also worked with Chyongere Hsieh in preparing specimens for high-pressure freezing. Tomographic reconstructions were made from thin, conventionally-prepared plastic sections provided by Dr. Franzini-Armstrong. This was done in order to learn to recognize the ideal orientation of the muscle fiber for revealing the DHPR. Initially, we are studying tomographic reconstructions from plastic sections. We have so far made six tomograms of plastic-embedded material supplied to us by Dr. Franzini-Armstrong, and we are analyzing this data for evidence of dihydropyridine receptors or other proteins related to excitation-contraction